Note: Descriptions are shown in the official language in which they were submitted.
755
SpEC99
BIOLpGICAL AGENT'FOR CONTROL OF CROP FUNGAL DISEASE
1
FIELD OF INVENTIpN
The present invention relates genera~.ly to a
biological control agent for certain crop fungal diseases.
More particularly, the present invention relates to a
bacterial strains of Bacil,_ lus megaterium de Nary which can be
used as biological control agents for soybean diseas~s ~ueh
as those caused by the fungus Rhizoctonia solani Ruhn. The
biological cantro~.,agent contemplated herein can also be used
1G for enhancement of soybean plant growth and yield, as.well as
for the production of antibiotics directed toward crop fungal
diseases.
BACKGROUND 4F THE INVEnTTION
15 The soybean, Glycine mar (L. ) i~ierr~.ll, was
domesticated by the farmers in the eastern half of northern
China during the Shang dynasty (ca. 1700.1100 B.C.), and
first introduced tv North America in 17f5 by Samuel 8owen, a
seaman employed by the East Tndia Company, from China via
London to Savannah (in the, colony of Georgia). He
manufactured soy sauce and e~cported it to London. In 1851,
soybeans were introduced into the midwestern U.S, by Dr.
Benjamin F. Edwards, who gave them to J.B. Lea, of Alton,
Illinois, who planted them in his qarden_ By 185, eoybearr~t
had been disseminated throughout.the United State,
World oilseed production for 1987-88 was estimated
nt ~1 ~:V~~:Vlll 2y,4, million zone, with soybeans accounting fox
ball the tocai crop. ~rne United Statea produced 51.8 million
tons of soybeans, representing over 51% of world production.
3Q Brazil had a record harvest of 18.5 million tons, and the
people's Republic of China harvested 11.8 million tons. The
fourth highest producer was Argentina with 8.5 million tons.
f
'Z- ~0~~'~3~
In 1987, the worldwide loss to soybean diseases was
1 estimated at 10.3 million metric tons. Losses in the United
States were estimated at 50-60 million dollars in 7,986
(estimates vary, depending upon market price). More than 100
pathogens are known to aff~ct soybeans; about 35 are
important economically. Same of the most important are those
that cause damage to the roots anti crowns of soybeans. One
ar um mast important ' rooL and crown rot aiseases are those
caused by Rhizoctonia solani. Rhizocton~.a diseases,
including pre- and post-emergence damping-off, root and stem
decay, and leaf and bud blight, have been reported ~.z~ all
soybean-growing areas of the world. Pre- and post~emergence
damping~off and root decay can reduce stands by as much as
50~ and losses of up to ~0$ have been recorded in Brazil and
the U.S. The causal fungus has a wide host range, which
includes field crops, vegetables, ornamentals, and fruit
crops.
Pre-emergence blight caused by _R. sod occurs
immediately after the seedling emerges from the seed. The
sprouted seed is killed and decayed by the cau$al fungus.
Damping~off can oCaur a few days after emerg~ance.
Reddish-brown lesions appear at the base of the seedling stern
and. on roots just below the soil line. These may enlarge
into sunken lesions, which may girdle the stem. Lesions and
cankers may so weaken the stem that p7.ant~ break off in
~ mid-season or die. Decay may continue intermittently
throughout the growing season, with continuing death of
plants. Infected plants tha~L su,rvi~cre s)mw scuc~e~ yi,~ld
reduction.
35
..3- .
2~~~'~E~
_R, sod
is primarily
a soil
inhabitant
and
has
1 excellent
saprophytic
ability.
The
fungus
can
overseason
in
the absence
of the
host,
arid
eolt~nizes
all
types
of plant
debris.
Growth
in soil
depend$
on nutrient
supply,
soil
moisture,
temperature,
pH,
and
competition
from
other
soil
microorganisms.
The
population
of the
fungus
generally
is
distributed
mainly
in the
upper
cm
(centimeters)
of the
soil,
deczeasing
with
depth
to about
50 cm
be~.c~w'the
:urfaae.
When
environmental
conditions
are
optimal
for
the
fungus,
disease
severity
i.s
directly
related
to inoGUlum
potential.
10 ~
inoculum
density
o 100
ug of
myas~.ia/gram
of soil
can
_ cause
severe
disease
in soybeans.
The
fungus
produces
pectolyt~ic
and
proteolytic
enzymes
which
play
a .role
is
disease
pathology.
There
is no
natural
resistance
to
~t. solani
in
_
1~ soybeans
ox any
other
crop.
An integrated
disease
m$~
~nt
program ,
has
been
used
heretofore
against
this
fungus
which
includes ,
agronvmia
practises
as well
as fungicid~a
seed
~
protectants.
The seed
protectant:
which
may
6e used
alone
or
in combination
with
other
fungicides
for
this
purpose
are=
q,~intozene
(pentachloronitrobenzene,
Terraclor)
or aarboxiri
(DCMO,
Vitavax).
t3oth,
but
particularly
quintoaene,
cause
delayed
emergence
and
growth
.of
ge~xdJ.ings.
An integrated
disease
management
program
is required,
which.
would
include
the use
of a
suppressive
b~.ologioal
control
agent.
Thr
present
~HV$ntion
detailR
rhP
~.,se
of such
an agoat,
and
in
pert
i rm
lar,
thQ
us~a
of particular
Dee~.llus'
~nt,~~
E.a~.~.~u
~. ~.L
dtilli
~ m
as biological
- .
control
agents
fox
R. solani.
_
..
The agricultural
use
of B,
m~ater,_
ium
has
beau
_ previously
xeported
for
disease
aoutrol
in rice~and
cotton.
3Q Inhibition
of Drechslera
aryzae,
which
cans~s
brown
Spo'~.
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disease'in rice, by B. megateri.um,~and subsequent control. of
the.disease was reported with regular spraying of a bacterial
suspension. In field studies, spraying w~i,th a suspension of .
- B. m~e~aterium reduced rice disease incidenoe and resulted in
bef.ter growth and higher rice yields, [Islam, K.Z., et _al. (2~85~
Z. Pflkrankh. Pflschutz iJourn$1 of Plant Diseases and ~'
Protection) 92:241»246]~. The use of a suspension of B..
me9at~ reduced the number of cotton plants killed by.~
P~matot~r~ichum omnivorum, cause of cotton root rot, bx 25%
'with a resulting yield increa:e in lint of 24% over the
aontro~ ~[Cook, C. G., et al. (1987), proc. Beltwide Cotton
Production - Mechanization Research Conf. Memphis, p. 4345:1
Antibiotic production from B. meaatexium has been
observed. Nerdy (CRC Handbook of Antibiotic Compounds, Vols.
I-XIV, CRC Press, Znc., scca Raton, FZ, 19$01987) reports
production of sueh antibiotics as ansamitocin-PDbi-p,
bacimethrin, megacin, pentapeptide, homopeptides. Thes~a are
prote~.de antibiotics having relatively lor~~ mammalian
toracity. Additionally, $. megaterium was, reported to
inhibit ,three fungal pathogens of 'the rise phyl,loplanG with' '
.2C the active fungicidal component being an antibiot~.c with a
lipopeptide: and polyoxin nature (Islam, K.z., t _al. (1985)
2. Pflkrankh and Pflsahutz 92:233-240].
. A variety of other uses for _e, meafaterium_ and its ,
metabolites have been reported, including o~eidation of
25; selenium, phosphate solubilizata.on, pxoduotxon of vitamin H12
in GQxn meal and the ability to degrade the herbicide
. metol.a~hlox . The use of any B . megatexium strains to inhibit .
funga;l':.,gxowth, disease or infeotipn in,soybeans has not been ~
~i'deserxbed ox::repaxted.
L. S
I 1 J.. . ..
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. . . . ' . ' . ~ ., . . ' : ~ ~ ~,~ .... ,....,
~''...','.::.,~." t' .~,'::,'.!'...~.. ' .:.~.'.::~:'~~~~;'~:.':~ ',.~~. .
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~p ~ 1r d ~ y y. ~.~, t ~ ~ '
t iFYr . ~ ~ l r .s'' ~ . . . '
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c ?i
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In laboratory culture studies, one _H. mec~aterium
strain was found to oxidize selenium to selenite and re trace
of selenate. This may be an important means of providing
sufficient selenium to herbage to ultimately prevent selenium
deficiency in animals [Sarathcha,ndra, S.U., et _al. (19$1)
Scir~ ence 2.1:600-601). A product, Phosphabacterin, from the
U.S.S.R. and containing cells of $. megatexium oar.
phosphaticum, was used in the Soviet Union and Eastern Europe
for the bacterization of crops to exploit the phosphate
solub~.7.izatfan properties of the bacterium, Additional
studies using the phosphate-solubilizing microorganisms on
various crops in field studies were reported in India
[Subba-~aQ, N.S., (7982) in Advances in A~ricu7.tural
Microbiology (N. S. Subbao-Rao, ed.) p. 219-242, Oxford Press,
New Delhi: Oxford]. Various _H, me_gaterium strains or
isolates have been reported to produce vitamin Blz in corn
meal [Chung, Vii. J. et al, (1986) J. Foofi Sci. 51:1519-157.7]
or to degrade metolachlor [Saxena, A. et al. (1989) Appl.
Environ. Microbial. 53:340-396].
SvMMA~Y of TxE zNVENTZON
The present invention providers biological agents
for control of certain fungal diseases found in soybeans and
other crops. Moreover, these agents can also stimulate
growth and yield in soybeans. Specifically, these agents are
5 particular strains o~ B. megaterium that exhibit the property
.of inhibiting fungal pathogens of soybeans in a
non-phytotoxic manner and may al$o produce one or more .
antibiotics. The use of these strains instead of current
seed and soil treatment fungicides allows reduction of
environmental contamination by such fungicides.
~5
~6r
Another aspect of the prexsent invention relnt~es to
1 a biological control agent which pzor~uces ena or more
antibiotics capable pf ~,nhihifiing fiung~l pathc7gAris of
- soybeans or other crops.
Still another aspect of the present invention is
directed to an improved method for soybean crop fungal
disease control and compositions useful in treating fungal
diseases.
BRIEF DESCRxPTION OF THE DRAtyIhG
Fig. 1 i$ a graphical representation of the
percentage reco~~ery of the fungal pathGgan R. solani
remaining in the soybean root rhizosphere after treatment
kith a a, meaaterium ~.salate (right panel, noted as aA) or
after no treatment (left panel, noted as R).
lj
DETAILED DESCRZPTIOr; OF THE INVENTION
The present invention relates to a biological agent
for the control of fungal disease in, soybeans. t~:are
specifically, the present invention relates to _B. meaaterium,
strains which can be used as control agents against diseases
cau$ed by' R. solani in soybeans. Such strains oz 8.
_ _
megaterium hcwe been isolated from a group of naturally-
occurring isolates of Bacillus, and do not require genetic
alteration for use as effective biological control agents.
The unexpected and surprising attributes of these
spec~.fic B. megaterium stxai.ns as biological control agents
include the following charaeterigtics. The strains have a ,
rapid generation ;t~.me in culture ( 28 minutes at . 3~ °C1 and a .
xifampicin~ resistant marker, .w~,~~,h . ;. , _ .
providesa means tt~ monitor the microorganism in the field or
_'_
. ~U3~'~~~
elsewhere. In pure culture these agents inhibit the growth
of most fungal pathogens of soybeans, without being
phytotoxic to soybeans or zoopathogenic. A pure culture or a
- cell-free culture supernatant of these B. mectaterium strains
can inhibit the vegetative growth of R. solani, a pathogen
which causes the Rhizoctonia diseases or soybean crowns,
roots, hypocotyis and sterc~s. The 8. megaterium strains of
the present invention have a rapid growth rate, are capable
of colonizing the soybean root system, can survive in the
soybean root rhizosphere, and can overwinter in the field at
temperatures as low as about 0 to about --10°C in the soil.
These bacteria reduce the diseases caused by R. sdlani in
soil both under greenhouse conditions and in the field.
Hioreover, the present strains continue to sLrvive in the soil
during the growing season and shown sustained antagonism to
the pathogen R. solani.
The subject 8. megaterium strains cr~n also
stimulate lateral root production on soybean plants under
controlled environmental conditions and in the field. Such
agents are further capable of stimulating the production of
nitrogen-fixing nodules on soybean plants in the field. The
nodules are typically produced by another bacterium,
Bradvrhizobium ~aponicum, but these agents further stimulate
the production of nodules, zn addition, these bacterial
strains appear to produce one or more antibiotics which
inhibit _R. solani yr other tungal.pathogena of soybeans sinoe
culture supernatants and cell-free culture filtrates exhibit
c_rrowth inhibitory effent~; fnr R. ~nl ,»i. ,
,The subject B. megaterium strains have the
taxonomic characteristic~c listed in ~~abte 1, as compareti to
those of B. subtilis and the prior art strains of H.
megaterium.
f'
. . -gw ~ 2P t~ Lt
TA.BLL 1
Comparisons of biochemical and physical charaaterisitcs of
Bac' illus me ateriur~ isolate 5153-2-~2 (invention) , ~sacillu$
meaateri.um (descriptive) and 8ac~ illus sub tills (descriptive).
CharaCtex'lStxG $Sc:;, lus maceter~,u~; 8ac~ Sllb
rnver.tion Descript.ivev DesCriptivev
~
Gram reaction ~- (y~) + +
Mo ti 1 ity + +
Catalase reaction + + +
' Anaerobic -(x) _
Voges-Proskauer (V-p) - .. +
Maximum temperature ~VC ~5C 55C
tiinimum temperature .SC 15C i5C
NaC3--5b ~ - 0 ( 'I +
-' ~
~;~cl-7 g + r +
i'TaCI-l0 ~ + - 0
Pdediun: c:I 5.7 + + +
Acid from glucose + + +
~~.gment ~~ ~<~r~Sl:Z~ '~ +~-(Z, r
Starch hydrolysis + + ~ +
Citrate utilization T + +
rropionate utilization + +
Nltl'at~ t0 nitrl.te 'f -f-~_ ,E
Casein decomposition + ~. +
Tyrosine decomposition + +/_ -
Litmus milk Alkaline Alkaline Alkaline
Lysvzyme + +
pH in V-P broth 7.5 4.5~G.8 6.5-7.0
Unstained globules + +
Cell width 1.2-1.3 um ~ 1 um ~ 1 um
v = As described ~.n: Cordon R.E., Halrii~,s, ~ w,C:,, anti riQx-Nay
Pang, C. 1973. The Genus Bacillus USDA, ARS Agr, Handbook No.
427, U.S. Dept., Agr, #tes. Sv., Washington, D.C,, X83 pp.
4 w =Positive reaction
x ~ negative react~.on
~y - not available
z = reaction variable
r
A represeratative isolate of the strains of the
present invention has been deposited with the American Type
Culture Collection, 12301 Parklawn Drive, Rockvzllc, rID
- 20852. This isolate is B. merLaterium strain 8153»2-2
assigned accession numbex ATCC 55000.
In addition tv the other properties noted above,
these novel B. megaterium strains also have the following
gualities which are important tar use as a biological
control agents for soybean crop root pathog~:ns by:
a) being a naturally-occurring isolate or str«in
of a nonplant-parasitic microorganism that does not require
genetic alteration to be effective,
b) being easily cultured ana having a rapid
generation time ir. culture,
c) exhibiting rapid initial growth rate in the
field,
d) having a stage in its life cyole that i$
resistant to harsh environmental conditions,
e) being suppressive to one or more soybean root
plant pathogens in the field,
C f) being relatively easy to develop for commercial
purposes,
g) reducing the population of a soybean root
pathogens) in the filed,
h) reducing symptoms of fungal diseases) on the
host soybean crop in the field,
i) colonizing the root ayste~m of the host soybean
plants) fox the pathogens) involved,
j? colonizing the rhizosphere of the root system
of the host soybean plant(s),
k) remaining in the soil for the full growing
season,
-~o~
2~~~'~ 3~
1) being non-phytotoxic to the host soybean
1 plant (s) .
m) enhancing the root growth of the host soybean
- plant(s).
n) enhancing the yield of the host soybean
plant (s) , and
o) not adverse~.y affecting nodule formation, in
the soybean plants, but rather promoting $radyrhizobium~
induced nodule formation in such plants.
Another aspect of this invention is directed to a
method oz controlling fungal diseases in soybean crape,
especially tc~ cvrtrolling diseases caused by _R. solani.
Crops axe treated pre~emergenc~ or poet-emergence of the
seedling ~;ith a fungicidally effectivr~ amount of the _B.
me aterium strains of the present invention. The _B.
1~ me aterium 8153-c-~ stxain designated ATCC 55000 is
especially preferred in this process.
The term "fungici.da~.ly effective" amount is defined
herein to be the population of the subject H. megaterium
strain inoculum required to reduce the pathological effect$
C of fungal pathogens and to obtain the desired populat~.c~n of
the subject B. megaterium strain in the soil and on the
plant.
Treatment on a pre-emergence basis includes
treatment of soybean seeds from any time prior to
implantation up to the appearance of a seedling.
Post-emergence treatment then enaompa.sses treatment after the
seedling appeaxs above the soil. ..
The present method may be used with soybean crops
or plants grown in the greenhouse or in the field. An
inoculant of the subject strains is used such that
~11-
colonization in the range of about 103-108 colony forming
~ units/gram (cfu/g) soil occurs and preferably about I04~10~
e~u/g. The inoculant can be applied directly to the seeds or
- plants, can be present in the soil before planting or can be
distributed, e.g, by spreading dusting or the like, over the
crap or soil where the crop has been planted.
Seeds can be treated by coating with a composition
containing the subject bacteria by dipping in a liquid
containing these bacteria, by spray~.n,g with the liquid, or
other method known in the art for applying bacteria to seeds.
when
present in peat, the bacteria are grown in
broth to the necessary amount, then concentrated and mixed
with, peat or soil at the desired inoculum. Optionally thfs
mixture may be cured by well known methods to farm a granular
peat composition,
The carriers that may be used to disperse the
subject strains on a pre or post-emergence basis would
inc~.ude all those commonly used for dispersing fungicides on
soybean crops and would'includ.e carriers such as water; clay,
talc and other dusting agents. The baoteria in such
.20 compositions are at a level of about 103-108 cfu/g carrier,
especially 10~-108 cfu/g or 10~~ I08 cfu/g, provided that
concentration is a fungicidally effective amount for the
compos~.tion in question.
Any of the above compositions, liquids, powders,
2~ peat, soil and the l~.ke may have nutrients included therein
or appropriate carrier medium such as water, oils ox solid
bases such as powders, peat, sail, clay, talc and any other -
ducting agents. .
The follow~.ng example serves to further illustrate
0 this inwer~txon without limiting the same.
A collection was made of Back species from
soybean leaves, stems, crowns, and roots, as well as from
_ soil of a field i.n soybean monoculture tr~r over 25 years.
Five B. me ater ~ isolates were obtained which were
antagonistic to R. soZ,arii, but not phy~.otoxic to soybeans.
ane H, me~a~ ~ isolate was selected from this group, for
further investigation of its properties. This isolate is the
B' me aterium 8153-2-2 strain designated ATCC 55000 and it
showed a strong ability to suppress R. salani and symptoms
caused by R. sod on soybeans in the greenhouse and the
field. .
This isolate has the above noted taxonomic
characteristics and in addition contains a rifampicin-
resistance marker so that its calvnization of soybean roots
l~ can be monitored in greenhouse and~field studies.
Seedling$ from soybean seeds treated with a
suspension of the isolate in water were grown in a greenhouse
in soil infected with R. solani and had a significantly
(P-0.05) lower disease index and number of lesions/seedling
.20 than those from untreated seers /Table ?.).
Field application of this isolate (hTCC 55000) in
Illinois showed a significantly improved st~ybean seedling
stand compared with untreated soybean seedlings infested with
R. solani. P,, sc~ was recovered significantly les$ often
from soil planted with soybean seeds treated with the isolat
a
than from soil planted with untreated soybean seeds as
illustrated in Fig, 1. The field application c~f the ATCC
55000 strain was carr~.ed out using a apmpos~.ti.on containing
about 10~~.zp$ c fu/g of soybean stem powder as a carrier. A
range of about 8 to 10 pounds of this composition can be
,applied per acre of crop area:
3S
2(~~~'~~~
Colonization of soybean roots by ATCC 55000
1 extended to 20 cm below and 5 cm above the soil line using
bacterium--coated soybean seeds. The highest populations of
the isolate found on the plant were about 1.2 x 108 cfu~g
fresh root .and occurxed at 5 em 6elo~,r the soil line. In the
field, this bacterium was recoverab~.e at $ distance of at
least .30 cm. horizonta~,ly anc et least 50 nm vertically from
the application site at about 3U to 40 days after
application. Maximum rhizosphere population recov~red was 4
x 10~ cfu~g soil, and 'the minimum rhizosphere populati4n
recovered was about ~.0~ cfu/g soil at about x.00 to 120 days
after application.
The population of this strain was high at the
. beginning of the season and decreased toward the ena of the
growing season. It remained stable and similar in population
to that of naturally--occurring ~sacillus species throughout
the winter.. A population base of about 8 ~: 10~ (~ lOly cfu/g
soil was needed for survivz~.. The presence ox~ absence of
R~ solani d.id not affect colonizt~tion on roots or ~in the
rhixosphere.
20 Root volume and dry weight df plants txeated with
the ATCC 55000 strain was significantly highex than those of
untreated plants or plants treated with ~, subs strain
CA8 (Table 2). There was also a significant increase in '
modulation by Bradyrhizobium '~~poniau~a obser~red in plants
2~ treated with this isolate. coil application resulted ~.n a _
significant increase in yield as casnpaxed to treatment With
R, solan~ alone or R. sod and thg ~,TCC 55000 strain.
35
-1~- 20~~'~~~
TALE 2
Co~p~rison of the biocontrol potential of D
. met =
isolate ATC C ''
55000 z
a
n against the
g, subtilis isolate
CA ~
soybean fun al
9 pathogen R. so soybea ns in
lani on
~ the
greenhouse.
Treatment Lesion Disease It. solari R
t
co Root Plant
- -----~._
no. index Recovery vo.I. wt. dry wt.
($) (~~ (ml) Img) (mg)
i0
R. ao~ 13.2 82.3 94
0
. 352.5 45
R. solani + 8 .6 193.1
* 9 *
/a
*
. 81.0 472.5 54.2 231
70.5 9
ATCC 55000 .
R. sod +. 9,9 71 b
0
. ~ 445.0 40.8 165.2
CA $
W5
a - Number followed by an asterisk is significantly diffsrent~P=o.0~~
compared with the treatment with R. solani alone.
b - No data.
2~
35
